The high affinity receptor for IgE ("E-receptor") on mast cells and basophils plays a central role in immediate hypersensitivity reactions. Reaction of receptor-bound IgE with polyvalent antigen clusters the receptors and this stimulates cellular secretion of both preformed and newly synthesized mediators of inflammation. Our studies focus on the molecular mechanisms by which aggregation of E-receptors generate these responses. The similarity of E-receptors to other receptors of the immune system (e.g. the clonotypic receptors on T and B lymphocytes), make it likely that the significance of such studies extends beyond the IgE/mast cell system. During the past year, our principal studies have been along five lines: 1) We utilized Chinese Hamster Ovary cells permanently transfected with E-receptors to analyze the receptors' interaction of Lyn kinase. The small amount of endogenous Src-family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of E-receptors but not after addition of dimers of IgE. Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced. On the other hand, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn. Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the beta chain but not with the receptor's other cytoplasmic domains. Studies have been initiated to utilize this transfection system further to characterize the molecular sites where the receptor and kinase interact as well as the role of specialized domains in the plasma membrane which may promote such interactions. 2) We have begun to test the hypothesis that ligand affinity should be positively correlated with the number of sequential perturbations that the E-receptors can stimulate. This formulation, dubbed kinetic proof reading by McKeithon, suggests that the natural history of a cell may be very different when triggered by ligands of differing affinity for the same receptor. 3) Substantial progress has been made in assessing the change in the profile of genes that are expressed in activated vs resting mast cells using Serial Analysis of Gene Expression (SAGE) methodology. Among the interesting findings are: a) up-regulation of genes not previously described; b) up-regulation of genes not previously known to be active in mast cells; and c) constitutive expression of genes not known to be active in mast cells. It is apparent that this is a powerful method for defining the complex genetic activity of a cell. 4) We are extending our characterization of the phosphatases that dephosphorylate activated receptors.